Transduction of many external stimuli (e.g. light, odorants, neurotransmitters, hormones) by cells in all tissues occurs through activation of members of a large gene superfamily called G-protein coupled receptors (GPCRs) that couple receptor activation to complex cascades of signaling events through a vast and expanding array of intracellular and membrane bound proteins. A common feature of the transduction process for GPCRs is the intermediation of heterotrimeric G-proteins. Upon agonist binding to GPCRs, dissociation or rearrangement of the cognate G1 and G23 subunits is thought to trigger stimulation or inhibition of various enzymes, ion channels, and other effector molecules. Classical receptor theory suggests that any agonist of a specific GPCR will induce the same conformational changes, and that differences in efficacy and potency among agonists reflect only the coupling efficiency (strength of signal) and relative binding affinity. Recent evidence for a number of GPCRs has alternatively suggested the existence of ligand-specific conformations that preferentially traffic receptor activation to select subsets of signaling pathways resulting in distinct signal cascades for different agonists through the same GPCR isoform. In the case of the human D2 dopamine receptor, we have discovered selective activation patterns (potency and intrinsic activity) for K and Ca channels, adenylyl cyclase, and MAP kinase upon exposure to the agonists quinpirole, DHX, and NPA. The underlying molecular mechanisms for this functional selectivity of agonist action are unknown. In the proposed research we will examine mechanisms underlying functional receptor-signal complexes in the neuroendocrine AtT20 cell line and primary rat neurons through four specific aims: (1) To define ligand- dependent selectivity of D2 and D3 dopamine receptors coupled to five effectors; adenylyl cyclase (AC), Kir3.0 channels, CaV2.0 channels, p42/44 MAP kinase (Erk1/2), and 2-arrestin translocation. (2) To assess the parameters of functional selectivity for native D2 receptor signaling in rat midbrain neurons. (3) To test the contribution of conserved residues in transmembrane domains (TM2, TM5, TM7) and intracellular loops previously shown to be signal switches in other GPCRs to functional selectivity of agonist signaling through D2 receptors. (4) To test the hypothesis that specific receptor/G1-protein complexes confer agonist-dependent functional selectivity. These experiments will increase understanding of the role of intermolecular interactions in specifying the potency and efficacy of dopaminergic ligands and, in turn, will have impact on the development of drugs targeted toward several neuropsychiatric and endocrine disorders. PUBLIC HEALTH RELEVANCE: This research explores the molecular basis for a novel signaling phenomenon, termed functional selectivity, by which different drugs and neurotransmitters can direct very different signaling outcomes through the same isoform of neurotransmitter receptor. We will focus on an important class of dopamine receptors widely implicated in substance abuse behaviors as well as several psychiatric disorders such as schizophrenia. As the receptors are important targets for pharmaco-therapies, the outcome of our research will guide the development of more refined and specific drugs for these disorders with fewer side effects.